This R01 project will advance our understanding of the brainstem neural circuits that generate and control breathing behavior in humans and all mammals. Breathing is an integral part of cardiopulmonary physiology and understanding its neural origins has significant implications for human health. Rhythmic breathing movements begin during embryonic development and emanate from coordinated activity in brainstem respiratory neurons. One key population of rhythm-generating neurons is contained in a site called the preBtzinger complex (preBtC). The discovery of the preBtC made possible many powerful experiments that could be performed in vitro, and led to our contemporary understanding of the neurophysiology of respiration. Nevertheless, critical questions remain unanswered. Given the heterogeneity of respiratory-related and non-respiratory neurons in the preBtC, can we discover which neurons are the key rhythm generators? If rhythmogenic neurons can be identified (and we argue that indeed they can), then can we ascertain the cellular, synaptic, and molecular-level mechanisms that underlie rhythm generation? Finally, the importance of peptidergic modulation of respiratory rhythm has been widely recognized in the past decade, but its underlying biophysical mechanisms remain incompletely understood. This project seeks answers to these specific questions by studying the preBtC in thin brainstem slice preparations in vitro. SPECIFIC AIM 1 will evaluate the cellular composition of the preBtC. Transgenic mouse models will be used to apply fluorescent tags to genetically distinct sub-populations, and then selectively and serially lesion them to test their respective roles in rhythmogenesis. SPECIFIC AIM 2 will examine the synaptic-dendritic active membrane properties that generate inspiratory-related bursts. SPECIFIC AIM 3 will investigate whether presynaptic depression of excitatory transmission contributes to burst termination. SPECIFIC AIM 4 is designed to complement SPECIFIC AIM 3 by examining the postsynaptic membrane properties that also act to terminate inspiratory bursts. Finally, SPECIFIC AIM 5 will determine the ion channels that underlie respiratory modulation by key neuropeptides (and other neuromessengers). The new knowledge acquired during this project will aid in the treatment and prophylaxis of breathing disorders that result from failures in the brain and central nervous system. Moreover, studying a measurable behavior like breathing under controlled in vitro conditions helps reveal important principles that link neurons, synapses, and molecules to full-scale physiological behaviors, which will be of great interest in neuroscience as well as cardiopulmonary physiology.